Specific Phosphorylation of a Site in the Full-Length Form of the α1 Subunit of the Cardiac L-Type Calcium Channel by Adenosine 3‘,5‘-Cyclic Monophosphate- Dependent Protein Kinase

Abstract

Voltage-gated L-type Ca2+ channels mediate Ca2+ entry into cells in response to membrane depolarization. Ca2+ entry through the cardiac Ca2+ channel determines the rate and force of contraction, and modulation of Ca2+ channel activity by beta-adrenergic agents acting through adenosine 3',5'-cyclic monophosphate-(cAMP)-dependent protein phosphorylation contributes to physiological regulation of cardiac function by the sympathetic nervous system. Immunoblotting experiments using site-directed anti-peptide antibodies against different peptide segments indicate that the alpha 1 subunit of the cardiac L-type Ca2+ channel exists in two size forms with apparent molecular masses of 240 and 210 kDa, which we call alpha 1(242) and alpha 1(210), Alpha 1(242) corresponds to the full-length cardiac alpha 1 subunit predicted from its cDNA sequence, while alpha 1(210) is truncated at its COOH terminus. Only alpha 1(242) is phosphorylated in vitro by cAMP-dependent protein kinase. Protein microsequencing and peptide mapping of wild-type and mutant fusion proteins show that this phosphorylation occurs at serine 1928 near the COOH terminus. Phosphorylation of this residue can be detected by phosphospecific antibodies raised against the corresponding phosphopeptide. Experiments with these antibodies show that alpha 1(242) is phosphorylated in intact cells expressing the cardiac alpha 1 subunit in response to increased intracellular levels of cAMP. These results identify serine 1928 on the alpha 1 subunit as a possible site of regulation by cAMP-dependent phosphorylation.

Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle

“…The predominant (Ͼ90%) short form results from in vivo proteolytic processing of the C terminus near amino acid residue 1685, approximately halfway through the C-terminal domain (14). Similarly, the related Ca V 1.2 channels in heart and brain are also truncated by in vivo proteolysis at a similar position in their C termini (17)(18)(19)(20). Surprisingly, the site of interaction with AKAP15 and PKA is located beyond the point of truncation in the C terminus (9).…”

β-Adrenergic–regulated phosphorylation of the skeletal muscle Ca V 1.1 channel in the fight-or-flight response

“…Numerous protein kinases have been shown to phosphorylate Ca V 1.1 channels in this laboratory and others by incubating the purified protein in vitro with protein kinases and [ 32 P]ATP and measuring incorporation of 32 P into the α 1 subunits (19,25,26,(38)(39)(40)(41)(42). By this approach, phosphorylation by PKA, PKC, PKG, and CaMKII has been measured, and specific sites of phosphorylation have been identified for PKA and other kinases.…”

“…Skeletal muscle Ca V 1.1 channels and cardiac Ca V 1.2 channels are both posttranslationally processed in vivo by proteolysis near the center of the 600-amino acid C-terminal domain (17)(18)(19)(20)(21)(22)(23), but proteolytic processing is not observed in transfected nonmuscle cells (24). The C terminus is a substrate for PKA, and the sites of most rapid phosphorylation of purified Ca V 1.1 and Ca V 1.2 in vitro are located in the distal C-terminal domain (19,25,26).…”

“…Skeletal muscle Ca V 1.1 channels and cardiac Ca V 1.2 channels are both posttranslationally processed in vivo by proteolysis near the center of the 600-amino acid C-terminal domain (17)(18)(19)(20)(21)(22)(23), but proteolytic processing is not observed in transfected nonmuscle cells (24). The C terminus is a substrate for PKA, and the sites of most rapid phosphorylation of purified Ca V 1.1 and Ca V 1.2 in vitro are located in the distal C-terminal domain (19,25,26). Despite proteolytic processing, the distal C terminus (dCT) beyond the point of proteolytic truncation is required for interaction with A Kinase Anchoring Protein 15 (AKAP15) (27)(28)(29), which in turn is required for β-adrenergic regulation of Ca V 1.1 and Ca V 1.2 channels in intact skeletal and cardiac myocytes (28)(29)(30)(31).…”

“…We have previously proposed that the autoinhibitory complex of the Ca V 1.2 channel with its proteolytically processed dCT noncovalently bound is the natural substrate for regulation by the β-adrenergic pathway, which relieves the autoinhibition by the dCT (24). Unfortunately, the only confirmed site of cAMP-dependent phosphorylation of the Ca V 1.2 channel [S1928 in the dCT (19,32)] is not conserved in Ca V 1.1 and is not required for regulation of Ca V 1.2 channels in vivo (33,34). Therefore, we set out to identify additional sites of cAMPdependent phosphorylation that may be responsible for regulation of Ca V 1.1 and Ca V 1.2 channels by the β-adrenergic pathway.…”